The predictors of lymphopenia and its effects on survival in locally advanced esophageal squamous cell carcinoma

ABSTRACT To investigate the impact of the effective radiation dose to immune cells (EDIC) and gross tumor volume (GTV) on lymphopenia and survival in patients with locally advanced esophageal squamous cell carcinoma (LAESCC). Between January 2013 and December 2020, 272 LAESCC patients were treated with definitive radiotherapy in two institutions. Based on radiation doses to the lungs, heart, and body region scanned, EDIC was calculated as an equal uniform dose to the total blood considering blood flow and fraction effect. The radiotherapy plan was used to calculate the GTVs. Lymphopenia was graded based on the lowest lymphocyte count during RT. The overall survival (OS), progress-free survival (PFS), and local recurrence-free survival (LRFS) were analyzed statistically. The lowest lymphocyte count was significantly correlated with EDIC (r= −0.389, p < .001) and GTV (r= −0.211, p < .001). Lymphopenia, EDIC, and GTV are risk factors for patients with ESCC. In a Kaplan-Meier analysis with EDIC and GTV as stratification factors, lymphopenia was not associated with OS in the EDIC>12.9 Gy group (p = .294)and EDIC ≤ 12.9 Gy group, and it was also not associated with OS in GTV>68.8 cm3 group (p = .242) and GTV ≤ 68.8 cm3 group(p = .165). GTV and EDIC had an impact on the relationship between lymphopenia and OS in patients with LAESCC undergoing definitive RT. Poorer OS, PFS, and LRFS are correlated with lymphopenia, higher EDIC, and larger GTV.


Introduction
The immune system is essential for preventing and curing cancer, 1,2 and peripheral blood lymphocytes are important immune system constituents. 3,4][7][8][9][10] Therefore, finding impact factors of lymphopenia can help to improve efficacy and choose high-risk ECC patients for treatment optimization and follow-up management.
However, most previous studies concentrated on adenocarcinoma and included patients at various stages of the disease.Furthermore, the lymphopenia cutoff values varied across studies.These factors may have influenced the outcome.As a result, we conducted this study to investigate the lymphopenia criteria and see if GTV and EDIC can influence lymphopenia and poor outcomes in patients with locally advanced esophageal squamous cell carcinoma (LAESCC).

Patients
Patients treated with definitive (chemo) radiotherapy (RT) at the radiation oncology department of the Second Affiliated Hospital and the Cancer Hospital of Guangxi Medical University between January 2013 and December 2020 were selected.272 patients were included with stage II-IVA (American Joint Committee on Cancer, eighth edition, cM1, positive nonregional lymph nodes and irradiated during radiotherapy).

Treatment
Patients received a total radiation dose of 50 to 70.4 Gy in 25-33 fractions with elective nodal irradiation.Radiation was administered for 5-7 weeks using IMRT.All patients received definitive RT with or without chemotherapy.The chemotherapy regimens were nedaplatin/cisplatin and paclitaxel/docetaxel/5-fluorouracil.

Data collection
The following information was gathered from medical records: age, gender, performance status, smoking history, drinking history, heart disease history, pathology, staging, differentiation, tumor location, treatment modality, and lymphocyte count.And the RT plan provided data for radiation dose, gross tumor volume (GTV), mean lung dose (MLD), mean heart dose (MHD), and mean body dose (MBD).
Lymphocyte counts (cells × 10 9 /L) were collected 1 week before the start of treatment, every week during RT, and 2 weeks after RT.Lymphopenia was graded based on the lowest lymphocyte count and the Common Terminology Criteria for Adverse Events, version 5.0.G4 nadir was defined as lymphocyte count<0.2× 10 9 /L.
The effective radiation dose to immune cells (EDIC) was calculated as follows using the model developed by Jin et al.- 11 and improved by Ladbury et al. 12 :

Statistical analysis
The primary endpoint of this study was Overall survival (OS) and the secondary endpoint was progression-free survival (PFS) and locoregional recurrence-free survival (LRFS).The survival duration was calculated from the start of treatment to the corresponding event (loco-regional recurrence, tumor progression, death, or the last follow-up).
To compare continuous and categorical variables, the independent sample t-test or the χ 2 test was used.According to receiving operating characteristic (ROC) survival curve analysis, the best cutoff value for EDIC and GTV was determined.The univariate and multivariate Cox regression models were used to investigate the relationship between survival and variables (inclusion criteria, p < .1).To examine correlations between variables, Spearman's rank correlation coefficient was utilized.Spearman's rank correlation coefficient was used to test correlations between variables.The tests were considered statistically significant if P < .05.All the p values were two-sided.SPSS 22.0 (IBM, Chicago, IL) and GraphPad Prism 9 were used for the statistical analyses.

Lymphopenia correlate with EDIC result in survival
The lowest lymphocyte count was correlated with EDIC (r = −0.389,p < .001).To investigate the relationship between lymphopenia and EDIC, the EDIC was divided into quartiles based on prevalence, and the percentage of G4 lymphopenia was 32.4% ( EDIC was used as a stratification factor in univariate analysis and Kaplan-Meier analysis to further investigate whether EDIC influences the prognostic role of lymphopenia on survival.After considering EDIC as a stratification factor, there was no discernible connection between lymphopenia and OS (HR = 1.184, p = .26)in univariate analysis.In Kaplan -Meier analysis, lymphopenia was not associated with OS in the EDIC>12.9Gy group (p = .294)(Figure 2a) and EDIC ≤12.9 Gy group (p = .637)(Figure 2b).Other survival statistics (PFS and LRFS) were list in Table 4.
There was no significant correlation between lymphopenia with OS after using GTV as a stratification factor (HR = 1.321, p = .055)in univariate analysis.In Kaplan Meier analysis, lymphopenia was not associated with OS in GTV>68.8cm 3 group (p = .242)(Figure 3a) and GTV ≤68.8 cm 3 group (p = .165)(Figure 3b).Other survival statistics (PFS and LRFS) were list in Table 4.

Discussion
In our study, we found that definitive RT for LAESCC can reduce the lymphocyte count, and whether concurrent chemotherapy has no discernible effect on the lymphocyte count.We also discovered that lymphopenia was linked to poor survival, which was influenced by GTV and EDIC.After adjusting for GTV and EDIC, lymphopenia's prognostic role during RT remained insignificant.The higher EDIC and larger GTV correlate with worse OS, PFS, and LRFS.][15] According to growing clinical data, low absolute lymphocyte count (ALC) during RT is associated with poor survival of ECC.A retrospective study 10 enrolled 504 patients with stage I-III ECC who received neoadjuvant or definitive CRT and discovered that G4 ALC nadir (defined as a nadir of < 200 cells/ml) was correlated with poor OS and disease-specific survival outcomes.The median OS in patients with G0-2 nadir and G4 nadir was significantly different (5.0 vs. 2.8 years, p = .027).It should be mentioned that the majority of the included patients were adenocarcinoma and received CRT with 50.4 Gy.To further investigate the role of ALC in patients with esophageal squamous cell carcinoma (ESCC)   treated with definitive RT, Wang et al. 16 conducted a study of 189 patients with ESCC treated with definitive RT (50-68 Gy) combined with or without chemotherapy.Patients were divided into two groups based on the cut-off value of ALC nadir (defined as 0.38 × 10 3 cells/µl), and there were significant differences in OS (p < .001),PFS (p = .0048),and LRFS (p < .0053) in low ALC nadir group (≤0.38 × 10 3 cells/µl) and high ALC nadir group(>0.38× 10 3 cells/µl).In our study, we also discovered that radiationrelated lymphopenia was linked to poor outcomes in patients with LAESCC.
Although the mechanisms underlying the relationship between lymphopenia and the outcome of ECC treatment are still unknown.However, a variety of studies have focused on the identification of risk factors for treatment-related lymphopenia in ECC.Xu et al. 17 improved the model used to calculate EDIC for ECC patients who received CRT and investigated the relationship between EDIC and lymphopenia.Higher EDIC values (>4 Gy) were associated with G4 nadir during CRT (67.3% vs. 40.8%,p < .001),and EDIC score was a significant risk factor for severe lymphopenia.EDIC was also an independent prognostic factor for OS, PFS, and distant metastasis-free survival (DMFS).Another study draws the same conclusion.Cai et al. 18 found that the cutoff value of EDIC was 10.3 Gy according to the ROC curve, then 146 patients with ECC were divided into the EDIC ≥10.3 Gy group and EDIC<10.3Gy group, and the final result showed that patients with EDIC ≥10.3 Gy had a significantly lower median OS (14.2 vs.39.6 months, p < .001),and PFS (28.1 vs.39.4 months, p = .024).It should be noted that they may disregard the impact of the other dosimetry parameters.There are certainly other studies focusing on this issue.Wang et al. 16 concluded that large PTVs affected a low ALC nadir during RT in the study mentioned above.In addition to PTVs, Xu et al. 19 showed that G4 nadir was related to other dosimetry parameters such as lung V10 and heart V10 in patients with ESCC treated with definitive CRT.And G0-3 nadir group was significantly associated with better OS (p < .001),PFS (p < .001),and DMFS (p = .008)than the G4 nadir group, but not with locoregional failure-free survival (LRFFS) (p = .202).Moreover, to look into the relationship between lymphopenia and radiation dose to marrow during CRT for ECC.Anderson et al. 20 calculated thoracic vertebra volume spared 5-40 Gy (TVS5-40) and found that TVS10-TVS40 was significantly associated with higher lymphocyte nadirs in 46 patients with ECC.Furthermore, Newman et al. 21investigated the relationship between lymphopenia and vertebra volume in 54 patients with ECC, finding that absolute vertebral volume receiving 10 Gy > 289 cc, 20 Gy ≥ 270 cc, and 30 Gy ≥ 197 cc all correlated with absolute lymphocyte nadir.More samples may be required to support this conclusion.Besides that, mixed pathology and stages may have an impact on the results.In our study, we included 272 LAESCC patients and discovered that lymphopenia does not correlate with OS if we used GTV or EDIC as a stratification factor based on the GTV and EDIC cutoff values.Then we concluded that GTV and EDIC affect the relationship between lymphopenia and OS.
Based on our findings, we can propose specific strategies to improve the outcomes.To begin, radiotherapy techniques can be improved to reduce the radiation dose delivered to bone marrow, circulating lymphocyte cells, the lung, and the heart.In addition, we can also identify high-risk patients and modify their treatment, such as induction chemotherapy for GTV reduction.Our study also has several limitations.At first, this was a retrospective study with selective bias.Second, there were limitations in the EDIC model.Third, other factors may correlate with lymphocyte counts, such as infections or medications.

Conclusions
In conclusion, GTV and EDIC had an impact on the relationship between lymphopenia and OS in patients with LAESCC undergoing definitive RT.Poorer OS, PFS, and LRFS are correlated with lymphopenia, higher EDIC, and larger GTV.'

Table 2 .
Univariable analysis of clinical and dosimetric variables with outcomes.

Table 3 .
Multivariate analysis of clinical and dosimetric variables with outcomes.

Table 4 .
Kaplan-Meier analysis of lymphopenia with outcomes.